This model was developed with the support of the National Institute of General Medical Sciences (NIGMS)/the National Institute of Health (NIH) grant 1P20GM103652 (Project# 3) (to MRA) and the American Heart Association (AHA) Grant-in-Aid 14GRNT20460291 (to MRA); the Brazilian government grant CAPES (to KR and FR); and a Brown University LINK award (to IM). We also acknowledge the outstanding technical support from our veterinarians and animal facility staff.

This animal protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Rhode Island Hospital.
1. Anesthesia and Intubation
<ol>
	<li>Weigh the mouse to calculate the dosage of post-operative pain medication.</li>
	<li>Place the mouse in an induction chamber and deliver 4% isoflurane for 9 - 10 min, monitoring the animal throughout. Turn on a hot bead sterilizer so that the apparatus can preheat to approximately 250 &#176;C. Preheating will take 15 - 20 min.</li>
	<li>Once the mouse reaches a deep plane of anesthesia, with a breathing rate of approximately 32 breaths/min, place the mouse supine on a Styrofoam board and use an elastic band secured under the top incisors to hold the mouth open. Confirm sedation by performing a toe pinch. Position a high-intensity illuminator above the mouse so that the oropharynx can be visualized.</li>
	<li>Use curved forceps to open the jaw and another pair of forceps to lift the tongue out of the way. Be sure to intubate while positioned at or slightly below eye-level with the body of the mouse. The use of surgical loupes is recommended.</li>
	<li>Visualize the opening and closing of the vocal cords. When open, insert a 20-gauge, 1-in intravenous (IV) catheter with a blunt-tip needle introducer. Use the needle to guide the catheter to the tracheal opening, but avoid inserting the needle into the trachea. Verification of correct placement can be done using a plastic transfer pipette.</li>
	<li>Transfer the intubated mouse to an operating surface equipped with a heating device. Connect the mouse to a small rodent ventilator set to a stroke volume of 150 &#181;L/stroke and a stroke rate of 130 strokes/min.</li>
	<li>Deliver 2.5% isoflurane. Verify the intubation by checking for bilateral chest rise. Verify anesthesia by performing a toe pinch. The mouse may need 5 - 10 min on the ventilator to become fully anesthetized.</li>
</ol>
2. Preparing the Mouse
<ol>
	<li>Tape down the intubation tube at the connecting site between the ventilator and the IV catheter. Tape down the extremities. Place sterile lubricating drops on the eyes.</li>
	<li>Trim the ventral left side of the thorax with an electric razor. Dust off the shaved fur with dry wipes and apply a small layer of hair removal cream using a sterile cotton swab. The cream should remain in contact with the hair follicles for approximately 30 - 45 s.</li>
	<li>While the cream processes, place three sterile cotton swabs in three 1.5-mL tubes filled with Betadine to soak. Using wipes moistened with distilled water, gently wipe away the cream and fur.</li>
	<li>Clean the surgical field three times, alternating Betadine and sterile 70% isopropanol prep pads, cleaning in a circular motion moving from center to periphery. Place a sterile drape with a quarter-sized hole over the surgical field of the mouse.</li>
	<li>Clean the area surrounding the mouse with 70% ethanol. Verify anesthesia once more with a toe pinch.</li>
</ol>
3. LAD Ligation
<ol>
	<li>Place the autoclaved surgical instruments in hot bead sterilizer preheated to 250 &#176;C for approximately 20 s. Place the sterilized instruments on sterile autoclaved surgical drape. Don surgical gloves.</li>
	<li>Use fine-tip forceps to gently lift the skin at a point approximately 5 mm to the left of the prominent xiphoid cartilage. Use a surgical scalpel with a No. 10 blade to create a vertical incision in the skin from this point upwards, to the level of the manubrium.</li>
	<li>Use curved forceps to gently separate the skin and muscle layers. Open the muscle layer, following the skin incision. Insert two 5-0 polypropylene sutures through the muscle layer, one on either side of the incision, and secure the sutures temporarily with clamps to hold the muscle layer open.</li>
	<li>Identify and make an incision in the third intercostal space, following the natural angle of the ribcage. Remove the tape from the left extremities of the mouse and secure its left rear foot to its right rear foot with tape. Cut a longer piece of tape and secure its left front foot to the operating surface in a slightly elevated position. Clean the gloves with 70% ethanol.</li>
	<li>Use a retractor to gently spread apart the 3rd and 4th ribs. Cut a small section of sterile gauze, approximately 1 in x &#189; in, and dip it in sterile 0.9% saline. Squeeze out the excess saline and use forceps to gently insert the gauze against the left lung to prevent accidental lung damage during the procedure.</li>
	<li>Gently remove the thin pericardium with forceps.</li>
	<li>Tear a small amount of cotton off a sterile cotton swab and roll it into a small ball. Dip this cotton ball into sterile 0.9% saline and gently swab over the surface of the heart to appreciate the arteries. Gently push the left auricle upwards and locate the coronary arteries underneath.</li>
	<li>Identify the LAD and pass an 8-0 nylon suture under the LAD; complete two throws to secure the ligation. If the ligation is successful, the left ventricle distal from the ligature will blanch.</li>
	<li>Using forceps, remove the gauze inserted earlier, and then gently remove the retractor. Insert a 6-in, 25-gauge flexible tube attached to a 25-gauge needle into the chest cavity through the thoracotomy opening. Advance approximately 1 - 2 in of tubing into the space above the left lung. Return the mouse to a supine position and clean the gloves with 70% ethanol.</li>
	<li>Use 5-0 polypropylene sutures in a simple interrupted pattern to close the ribcage, keeping the chest tube in place. Remove the two sutures holding the muscle layer open. Use 5-0 polypropylene sutures in a simple continuous pattern to close the muscle layer, again keeping the chest tube in place.</li>
	<li>Attach a 1-mL syringe to the 25-gauge needle on the chest tube. Gently pull upwards on the plunger while simultaneously gradually extracting the chest tube from the chest cavity with forceps. Extract the tubing slowly, as this step removes excess air and blood, which would otherwise become trapped in the chest cavity and result in a pneumothorax.</li>
	<li>Once the syringe is full, detach the syringe from the needle and dispose of the waste in a waste beaker or sink. Continue this process until the chest tube is completely extracted. Ensure that the chest is tightly sealed.</li>
	<li>Decrease the isoflurane to 1.5%. Close the skin with 4-0 polypropylene sutures in a simple interrupted pattern. Turn the isoflurane vaporizer off.</li>
	<li>Administer 0.1 mg/mL buprenorphine in 0.9% saline via an intraperitoneal (IP) injection. Topically apply 2 mg/mL Lidocaine with 2 mg/mL Bupivacaine in 0.9% saline to the incision. Administer between 200 - 500 &#181;L of 0.9% saline via a subcutaneous injection, scaling the saline amount to the weight of the mouse.</li>
	<li>Wait 5 min after administering the pain medications to remove the mouse from the intubation tube. This aids in the transition off the ventilator.
	<ol>
		<li>If the mouse does not have a bilateral chest rise once off the ventilator, perform needle decompression. To do this, introduce a 25-gauge sterile needle and a 1-mL syringe between the 3rd and 4th ribs until it enters the thoracic cavity, denoted by a sudden decrease in resistance. Pull up gently on the plunger to remove excess air.</li>
	</ol>
	</li>
	<li>When the mouse demonstrates an adequate bilateral breathing rate and depth and responds to a toe pinch, place the mouse in a clean recovery cage under a heat lamp. Provide the mouse with moist food and a water bottle, monitoring in a laminar flow hood for 15 - 20 min. Monitor for an exaggerated breathing effort, excessive bleeding, or other potentially life-threatening complications.</li>
	<li>For the next three days, administer 0.1 mg/mL buprenorphine pain medication via an IP injection twice daily. Monitor the mouse daily.</li>
</ol>

<ol>
	<li>Hausenloy, D. J., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+directions+for+protecting+the+heart+against+ischaemia-reperfusion+injury:+targeting+the+Reperfusion+Injury+Salvage+Kinase+(RISK)-pathway.">New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway.</a> Cardiovasc Res. 61 (3), 448-460 (2004).</li><li>Roffi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2015+ESC+Guidelines+for+the+management+of+acute+coronary+syndromes+in+patients+presenting+without+persistent+ST-segment+elevation.">2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation.</a> Eur Heart J. 37 (3), 267-315 (2015).</li><li>Kumar, A., Cannon, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Coronary+Syndromes:+Diagnosis+and+Management,+Part+I.">Acute Coronary Syndromes: Diagnosis and Management, Part I.</a> Mayo Clin Proc. 84 (10), 917-938 (2009).</li><li>Eitan, A., Nikolsky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithrombotic+therapy+in+patients+with+acute+coronary+syndromes:+how+to+make+the+right+choice.">Antithrombotic therapy in patients with acute coronary syndromes: how to make the right choice.</a> Minerva Med. 104 (4), 357-381 (2013).</li><li>Abbate, A., Bussani, R., Amin, M. S., Vetrovec, G. W., Baldi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction+and+heart+failure:+role+of+apoptosis.">Acute myocardial infarction and heart failure: role of apoptosis.</a> Int J Biochem Cell Biol. 38 (11), 1834-1840 (2006).</li><li>Zheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nebivolol+protects+against+myocardial+infarction+injury+via+stimulation+of+beta+3-adrenergic+receptors+and+nitric+oxide+signaling.">Nebivolol protects against myocardial infarction injury via stimulation of beta 3-adrenergic receptors and nitric oxide signaling.</a> PLOS ONE. 9 (5), 98179 (2014).</li><li>Tarnavski, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cardiac+surgery:+comprehensive+techniques+for+the+generation+of+mouse+models+of+human+diseases+and+their+application+for+genomic+studies.">Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies.</a> Physiol Genomics. 16 (3), 349-360 (2004).</li><li>Buitrago, S., Martin, T. E., Tetens-Woodring, J., Belicha-Villanueva, A., Wilding, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+Efficacy+of+Various+Combinations+of+Injectable+Anesthetics+in+BALB/c+Mice.">Safety and Efficacy of Various Combinations of Injectable Anesthetics in BALB/c Mice.</a> J Am Assoc Lab Anim Sci. 47 (1), 11-17 (2008).</li><li>Kolk, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAD-Ligation:+A+Murine+Model+of+Myocardial+Infarction.">LAD-Ligation: A Murine Model of Myocardial Infarction.</a> J Vis Exp. (32), e1438 (2009).</li><li>Wu, Y., Yin, X., Wijaya, C., Huang, M. H., McConnell, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction+in+rats.">Acute myocardial infarction in rats.</a> Journal of visualized experiments : J Vis Exp. (48), (2011).</li><li>Ferrera, R., Benhabbouche, S., Bopassa, J. C., Li, B., Ovize, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+hour+reperfusion+is+enough+to+assess+function+and+infarct+size+with+TTC+staining+in+Langendorff+rat+model.">One hour reperfusion is enough to assess function and infarct size with TTC staining in Langendorff rat model.</a> Cardiovasc Drugs Ther. 23 (4), 327-331 (2009).</li><li>Zeng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+5-ethynyl-2'-deoxyuridine+staining+as+a+sensitive+and+reliable+method+for+studying+cell+proliferation+in+the+adult+nervous+system.">Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.</a> Brain Res. 1319, 21-32 (2010).</li></ol>

The authors declare that they have no competing financial interests.

With an increasing use of the MI model in laboratories, the described procedure seeks to increase the efficiency and survival rate of the mice while minimizing their post-operative pain and discomfort. This protocol strives to minimize mortality by making numerous improvements to various aspects of the LAD ligation procedure. There are a few distinctions. Some murine intubation studies that utilize ketamine and xylazine along with isoflurane for induction, due to the benefit of their longer duration of anesthesia, have shown increased mortality8. Our method uses only isoflurane for induction, greatly reducing the potential for drug-related complications. Another similar LAD ligation protocol includes a tracheotomy, producing an extended recovery time and an increased need for precise training9. The procedure we describe here utilizes a non-invasive method of endotracheal intubation, resulting in lower mortality and higher reproducibility of results. Also, rather than only using an electric razor, our procedure also utilizes hair removal cream for fur removal, providing a completely clear visualization of the sterile field in less than 1 min.
Another key distinction is the repositioning of the mouse, which occurs after, rather than before, the initial incisions. Making the incisions while the mouse is supine allows for a more direct and accurate visualization of landmarks such as the xiphoid cartilage, thus resulting in higher reproducibility of results. Our method also uses sterile cotton swabs rather than a cauter for bleeding management, decreasing iatrogenic burn injuries and the risk of infection. Apart from these differences, the chest tube insertion for the thoracentesis is especially of note, as our method does not involve creating a new incision for the tube. Rather, it involves inserting the tube into a previous incision, again decreasing mortality. The procedure we describe also includes: (1) the use of a retractor, enabling a more accurate and stable visualization of the coronary arteries; (2) the insertion of sterile gauze into the chest cavity during the procedure, thereby reducing the risk of iatrogenic lung injuries; and (3) the administration of saline following the procedure, which has been shown to both shorten the recovery time and prevent hypothermia.
Although we describe a permanent ligation model, this procedure can also be modified for an acute MI model. The acute MI model, also described as ischemia and reperfusion, refers to 30 - 60 min of ischemia followed by reperfusion in the heart tissue7. An alternative method to assess the infarct size or the area at risk after ischemia and reperfusion is the staining of 2% triphenyl tetrazolium chloride (TTC)10. TTC staining is based on the ability to stain viable tissue after ischemic insult due to dehydrogenases present in cardiac tissue. These enzymes convert a soluble component into an insoluble red component, thereby delineating the infarcted region11. The acute MI model can mimic the mechanisms that occur in human heart disease and thus may be a useful tool for elucidating the events of myocardial ischemia10. The LAD ligation can be verified by observing an immediate change in tissue color, providing an advantage for this method of inducing an MI. Another method to check for successful ligation is the use of electrocardiograms, although this involves the use of expensive equipment and may not be feasible for all labs.
As described above, there are several easy and affordable molecular techniques to confirm the ligation after harvesting the heart. The two techniques shown above are Evan&#39;s Blue staining and trichrome staining. Evan&#39;s Blue dye is directly injected into the arch of the aorta, indicating where there is a lack of blood flow. This is a quick and efficient method employed immediately after harvesting the heart to test whether the model was successful and to measure the extent of coronary blockage. For trichrome staining, the heart must be sectioned and then subjected to immunohistochemistry. Trichrome staining can indicate post-ischemic fibrotic areas or cardiac areas affected by chronic ischemia. The injection of post-operative mice 2 - 24 h prior to sacrifice with 5-ethynyl-2&#8242;-deoxyuridine (EdU), an analogue for thymidine, is an effective method for indicating areas of DNA replication and cell proliferation following ischemia, especially in studies involving vascular regeneration12.
In general, the limitations of the LAD ligation study include the incidence of post-operative mortality, caused mainly by the presence of cardiac arrhythmias, bleeding, and pneumothorax. An effective thoracentesis, without additional chest incisions (described in the current method), and proper post-operative care are necessary to avoid morbidity and mortality in the animals. The very careful monitoring of post-operative hypothermia is also critical. The reduction in the number of neck and chest incisions (for tracheotomy and thoracentesis) described in the current method will help improve survival rates. The avoidance of injectable pre-operative anesthetics described herein will also improve the post-operative recovery of the animals.
In order to obtain high reproducibility, the LAD ligation model requires rigorous training and experience. The operator needs to perform several weeks of surgeries to gain the ability to reproducibly make infarcts at the desired sites on the heart. Training and experience are two critical factors for a successful LAD ligation survival surgery.

Coronary disease, or ACS, is the most prevalent cardiovascular event and will be considered the main cause of morbidity and mortality worldwide in 20201. The cause of ACS is the presence of a myocardial thrombosis due to the rupture of a coronary atherosclerotic plaque that blocks or reduces blood flow to the heart tissue2. Therefore, there are clinical signs consistent with the presence of acute myocardial ischemia, such as myocardial infarction (MI)3,4. MI leads to a loss in mass of the cardiomyocytes and a progression to pathological ventricular remodeling, which can lead to ventricular dysfunction and heart failure5,6.
One of the most effective ways to study IHD has been to mimic human myocardial infarction in an animal model. This is achieved by occluding the LAD in mice. Using this model, we study how the heart can be protected from the damage resulting from IHD.
Over the last decade, researchers have shifted from using larger animal models to smaller animals, including the shift from rats to mice. The smaller mouse model is starting to be preferred for many reasons, including their small size, large litter size, low cost to maintain, and short gestation period, as well as for the expansive availability of transgenic and gene knockout models7. Although mice are small in size, new surgical instruments specifically designed for them have aided in this development. Our method utilizes these new surgical instruments.
While several methods implement an invasive tracheotomy, we use a less invasive method of endotracheal intubation. Using overhead illumination of the oropharynx, we intubate without creating any incisions, providing a safer and less traumatic experience for the animal. The mouse is then placed on a ventilator and kept on isoflurane during the entire procedure. Due to the short duration of anesthesia produced by the drug, it only takes a few minutes for the animal to recover from the anesthetic once it is discontinued. Our surgical model also includes a minimally invasive thoracentesis. The careful removal of blood and excess air from the chest cavity using thoracentesis through the original thoracotomy incision has addressed a common post-operative complication of the LAD ligation: the tension pneumothorax. This method, which eliminates the need for the two additional incisions used in other methods-one for the tracheotomy and another for the thoracentesis-has yielded fewer post-surgical complications and has drastically reduced mortality.

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murine left anterior descending (lad) coronary artery ligation: an improved and simplified model for myocardial infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

The mice are euthanized twenty-eight days after surgery, and the hearts are harvested and examined. The mice are anesthetized with 50 - 75 mg/kg ketamine and 5 - 10 mg/kg xylazine. When the animal is under adequate anesthesia, the thoracic cavity is opened, and using a 23-gauge needle, cold potassium chloride (KCL, 30 mM) is injected into the posterior basal region of the heart. The heart is arrested in diastole. For further validation of the ligation, the heart is removed from the animal and is injected with 4% paraformaldehyde and then 1% Evan&#39;s Blue dye. Figure 1 shows the lack of Evan&#39;s Blue in the ischemic left ventricle. Figure 2 demonstrates a proper endotracheal intubation setup. Figure 3 displays the placement of the chest tube for the thoracentesis in the initial incision site, with the muscle layer sutured closed around the tube before air extraction from the chest cavity. Trichrome staining shows an increase in collagen in the infarcted region (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/55353/55353fig1.jpg" /> 
Figure 1: Evan&#39;s Blue Injection. The Evan&#39;s Blue injection reveals a lack of dye in ischemic tissue, localized to the left ventricle infarcted region. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55353/55353fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55353/55353fig2.jpg" /> 
Figure 2: Endotracheal Intubation. Demonstration of a proper endotracheal intubation setup. The operator, wearing surgical loupes, is seated at eye-level with the mouse. A high-intensity illuminator is focused downward onto the tracheal region, transilluminating the oropharynx. An elastic band is hooked behind the upper incisors, allowing the operator to open the mouth with the curved forceps. The curved forceps are used to hold the tongue to the side for clear visualization. An intravenous catheter intubation cannula with a blunt-tip needle introducer is advanced at a slight upward angle while the opening and closing of the vocal cords is visualized. Visualization of the vocal cords opening and closing before the intubation attempt is one of the critical points for a successful intubation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55353/55353fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55353/55353fig3.jpg" /> 
Figure 3: Thoracentesis. Placement of the chest tube used for thoracentesis, inserted in the original incision site. The muscle layer is sutured closed around the tube before the air within the chest cavity is extracted with a syringe, and then the chest tube is removed. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55353/55353fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55353/55353fig4.jpg" /> 
Figure 4: Trichrome Staining. Left panel: Control, non-LAD-ligated heart. Right panel: LAD-ligated, infarcted heart. Trichrome stain (Masson) employing Biebrich scarlet-acid fuchsin solution, phosphotungstic/phosphomolybdic acid solution, and aniline blue reveals increased collagen (blue) as a marker for fibrosis in the cross-sectioned left ventricle infarcted region. Bar = 500 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55353/55353fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction at JoVE.com

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

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24.3K Views.  Rhode Island Hospital, Brown University Warren Alpert Medical School. We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Video: Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...